Adeno-associated virus (AAV) is considered one of the most promising viral vectors for human gene therapy. AAV has the ability to efficiently infect dividing as well as non-dividing human cells, the AAV viral genome integrates into a single chromosomal site in the host cell's genome, and most importantly, even though AAV is present in many humans, it has never been associated with any disease. In view of these advantages, recombinant adeno-associated virus (rAAV) is being evaluated in gene therapy clinical trials, inter alia, for hemophilia B, malignant melanoma, cystic fibrosis.
Host cells that sustain AAV replication in vitro are all derived from mammalian cell types. Therefore, rAAV for use in gene therapy has traditionally been produced on mammalian cell lines such as e.g. 293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines. However, in most mammalian cell culture systems, the number of AAV particles generated per cell is of the order of 104 particles (reviewed in Clark, 2002, Kidney Int. 61(Suppl. 1): 9-15). For a clinical study, more than 1015 particles of rAAV may be required. To produce this number of rAAV particles, transfection and culture with approximately 1011 cultured human 293 cells, the equivalent of 5,000 175-cm2 flasks of cells, would be required, which means transfecting up to 1011 293 cells. Therefore, large scale production of rAAV using mammalian cell culture systems to obtain material for clinical trials has already proven to be problematic, production at commercial scale may not even be feasible. Furthermore there is always the risk, that a vector for clinical use that is produced in a mammalian cell culture will be contaminated with undesirable, perhaps pathogenic, material present in the mammalian host cell.
To overcome these problems of mammalian productions systems, recently, an AAV production system has been developed using insect cells (Urabe et al., 2002, Hum. Gene Ther. 13: 1935-1943; US 20030148506 and US 20040197895). This baculovirus expression vector system (BEVS) is based on infection of insect cells with baculoviruses containing a gene to be expressed flanked by AAV ITRs, a baculovirus expressing the AAV rep gene and a baculovirus encoding the AAV cap gene leading to production of infectious rAAV particles. If desired, the AAV rep and cap genes may be present on the same baculovirus
However, despite various improvements to the basic system, it is still problematic that more capsids appear to be empty rather than being loaded with the therapeutic gene of interest. There is thus still a need to overcome this limitation so as to improve large scale (commercial) production of AAV vectors in insect cells. Thus it is an object of the present invention to provide for means and methods that allow for stable and high yield (large scale) production of AAV vectors in insect cells.